Project Summary/Abstract Toxin-antitoxin (TA) systems are important for the ability of many bacteria to respond to environmental stress. These systems typically consist of a stable toxin that inhibits cell growth and an antitoxin that binds and sequesters its cognate toxin. Degradation of the antitoxin can thus liberate the toxin, providing an important mechanism of bacterial growth regulation. TA systems are prevalent across nearly all bacterial species, with many species harboring many copies, often as a result of extensive gene duplication. Despite their abundance, however, there is evidence that toxin- antitoxin interactions are remarkably insulated from each other; i.e. only co-operonic toxins and antitoxins have high-affinity interactions. This suggests that new TA systems deriving from gene duplications must rapidly diverge from one another to ensure that each toxin-antitoxin pair has a unique interaction specificity. How this divergence occurs at the molecular level remains poorly understood, and is crucial to understanding the expansion and prevalence of TA systems in bacteria. Here, I propose to characterize the effect of gene duplications on toxin-antitoxin interactions through study of the VapBC family of TA systems in M. tuberculosis, which has undergone a massive expansion through successive gene/operon duplication events. I will first characterize the interactions between toxins and antitoxins from paralogous M. tuberculosis VapBC systems to determine whether these systems are fully insulated from one another. I will then identify the specific amino acid substitutions responsible for a divergence in binding specificity for paralogous VapBC systems. Finally, I will determine the evolutionary trajectory followed by two VapBC systems post-duplication through the resurrection and characterization of ancestral proteins as well as evolutionary intermediates. Because TA systems are crucial for many bacteria to adopt dormant, persister states in response to stress, understanding the large expansion of TA systems that took place in M. tuberculosis will help illuminate the molecular changes required for the evolution of pathogenicity in this species. Additionally, this work will shed important light on the general mechanisms governing the evolution of new protein-protein interactions.